L-glutamine-containing Dulbecco's Modified Eagle Medium (DMEM), penicillin/streptomycin and fetal bovine serum (FBS) were purchased from EuroClone SpA. Both phosphate buffer saline and trypsin were purchased from Thermo Fisher Scientific, Inc. Ethyl acetate was purchased from Sigma-Aldrich; Merck KGaA. The MTT reagent the stop solution [dimethyl sulfoxide (DMSO)] and trypan blue dye were provided by Promega Corporation. Flow tubes (BD Biosciences) were used for centrifugation. BD FACSDiva™ software (version 8.0; BD Biosciences) and StemPro™ Accutase™ Cell Dissociation Reagent (Gibco; Thermo Fisher Scientific, Inc.) were used in the present study.

In the present study, AgNPs were synthesized using Aspergillus flavus strain MG973280, which was obtain from American Type Culture Collection (ATCC). The fungal culture was prepared as previously described (36). Briefly, Aspergillus flavus spores were adjusted to a concentration of 2.0x10⁶ and cultivated in a complex broth medium that included 10 g/l glucose, 10 g/l yeast extract and 5 g/l NaCl (pH 7). Subsequently, the culture was incubated for 72 h at 33˚C and cells were agitated using an orbital shaker at 0.10 x g. Whatman^® grade 1 filter paper was used to filter the culture at the end of the incubation period. The resultant mycelia, or biomass, were then collected and washed with deionized distilled water.

To produce AgNPs using the fungal biomass, the method reported in the study by Jadidev and Narasimha (37) was followed with minor adjustments. Briefly, 10 g of the first crude biomass were agitated using an orbital shaker at 0.10 x g for 72 h while submerged in 100 ml of sterile deionized water at 33˚C and pH 7.0. After the crude biomass was filtered using Whatman^® grade 1 filter paper, the resulting suspension containing the fungal filtrate without biomass was collected. Subsequently, 100 ml of this biomass-free fungal filtrate was combined with 1 mM of silver nitrate to produce AgNPs. The mixtures were then continuously stirred in the dark at 27˚C for 72 h using a magnetic stirrer.

NPs were lyophilized at -54˚C and 0.2 mbar for 24 h using a Lyovapor L-200 freeze dryer (BUCHI UK Ltd.). Scanning electron microscope (SEM) images were obtained at 200 kV using a JEM-2010 microscope (JEOL, Ltd.) in order to analyze the surface morphology of the AgNPs. The powdered samples were coated with gold using sputter coater and a carbon thread coater (Leica Biosystems). The resulting particle size was measured using the Zetasizer Nano ZSP (Malvern Panalytical, Ltd.).

Sponge samples were gathered from several locations in the Gulf of Aqaba (29˚27' N, 34˚58' E) by specialists from the Marine Science Station at the University of Jordan (Aqaba, Jordan). The sponges were found by divers between the depths of 1-18 meters. After being cleared of debris, the samples were frozen at -80˚C, and then sent in sealed sterile polyethylene containers (which were submerged in seawater in order to maintain a moist environment during transport) to Aqaba International Laboratories-BEN HAYYAN for freeze-drying and extraction. Using specified morphological traits listed in the Systema Porifera and World Porifera Database (35,38-42), the sponges, Stylissa carteri and Amphimedon chloros, were identified.

Sponge fragments were weighed, freeze-dried for 48 h at -40˚C and then ground into a powder. The powder (weighing 45.0-350.0 g) was soaked at room temperature for 48 h in a 1:1 mixture of methanol and dichloromethane. Subsequently, a lyophilizer was used to filter and dehydrate the solution. The final pure extract was divided into non-polar, semi-polar and polar components by dispersing it in distilled water and using n-hexane and ethyl acetate as partitioning agents. The pure extract from each solvent was labeled and kept frozen at -20˚C (43,44).

In the present study, to investigate the anticancer activity of the candidate mixtures, four cancer cell lines were used: i) A human lung cancer cell line (A549; cat. no. CCL-185; ATCC); ii) a human colorectal cancer cell line (HT-29; cat. no. HTB-38; ATCC); iii) a human breast cancer cell line (MCF7; cat. no. HTB-22; ATCC); and iv) a pancreatic cancer cell line (PANC-1; cat. no. CRL-1469; ATCC). For selective purposes, a normal human umbilical vein endothelial cell line (HUVEC; cat. no. CRL-1730; ATCC) was used. All of the cell lines, were cultured in DMEM containing 10% FBS, 10 mM HEPES buffer, 100 µg/ml L-glutamine, 50 µg/ml gentamicin, 100 µg/ml penicillin and 100 mg/ml streptomycin. ATCC provided authenticated cell lines, which ensured that the cell lines were correctly identified and contamination-free. Additionally, mycoplasma testing was performed on all of the cell lines used in the present study, which confirmed the absence of mycoplasma contamination.

A colorimetric MTT assay was used to evaluate the viability of the cells. Each type of cell line was seeded in 96-well plates at a density of 1x10⁴ cells per well and cultured for 24 h at 37˚C (45). Subsequently, the cells were treated with: i) The extracts of each of the sponge species, Stylissa carteri and Amphimedon chloros, separately at concentrations ranging from 6-200 µg/ml; ii) AgNPs at concentrations ranging from 0.75-200 µg/ml; or iii) mixtures of 0.75 µg/ml AgNPs with extracts of Stylissa carteri or Amphimedon chloros at concentrations ranging from 6-200 µg/ml. Following a 72-h incubation period at 37˚C (46), 15 µl of MTT solution was added to each well, and then the plates were incubated at 37˚C for 4 h. Subsequently, 100 µl of DMSO was added to each well to dissolve the formed formazan crystal. The BioTek ELx800™ microplate reader was then used to measure the optical density at 590/630 nm to assess cell growth.

LC-MS is an analytical method that combines MS and LC. LC separates components of a mixture by passing them through a chromatographic column. Even when LC is unable to positively identify these separated components, MS can be used to identify both known and unknown chemicals and provide information on their structures (47).

A mobile phase comprising solvents A and B in a gradient was used in the LC-MS analysis. Solvent A consisted of formic acid dissolved in water at a concentration of 0.1% (v/v), and solvent B consisted of formic acid dissolved in acetonitrile at the same concentration. The experimental parameters used included an Agilent Zorbax Eclipse XDB-C18 column (Agilent Technologies, Inc.) measuring 2.1x150 mm x3.5 µm, a temperature maintained at 25˚C, the sponge extract dissolved in methanol at a concentration of 18 mg/ml and an injection volume of 1 µl. The sample was injectedinto the LC-MS system, which included the following components: A mass detector using a SIL-30AC autosampler with a cooler, a Shimadzu CBM-20A system controller, an LC-30AD pump, a CTO-30 column oven and an electrospray ion-mass spectrometer with a skimmer voltage of 65 V and a fragmentor voltage of 125 V, all components were of the Shimadzu LC-MS 8030 (Shimadzu Corporation). Nitrogen gas with 99.99% purity and a flow rate of 10 l/min served as the drying gas during operation in positive ion mode. Additionally, a nebulizer operating at a pressure of 45 psi and a capillary temperature of 350˚C were used. Subsequently, the mass/number of ions of the eluent was scanned from positions 100-1,000. Authentic standard substances were used for result validation.

Molecular docking simulations were performed using tyrosine kinase receptor A [TrkA; Protein Data Bank (PDB) ID, 7VKO; https://www.rcsb.org/structure/7VKO] (48) and an EGFR kinase domain (PDB ID, 4I23; https://www.rcsb.org/structure/4i23) (49) in order to investigate the binding affinities of the major components in Stylissa carteri or Amphimedon chloros. AutoDock (version 4.2.6) (50,51) was used according the methods described in the studies by Saqallah et al (52) and Shtaiwi et al (53) with slight modifications. Briefly, all protein structures were prepared using BIOVIA^® Discovery Studio^® (version 16.1) (54) by removing water molecules (if applicable) and complexed co-structures. Complexed inhibitors (dacomitinib and repotrectinib in the EGFR kinase domain and TrkA structures, respectively) were separated from the crystal structures to be used as control ligands. Using AutoDockTools (version 1.5.6) (50), Kollman charges and polar hydrogen atoms were assigned to the proteins. Additionally, the 3D conformers of the compounds identified in the sponge extracts were downloaded from the NCBI PubChem database (pubchem.ncbi.nlm.nih.gov) and Gasteiger charges were assigned accordingly. A grid box with the size of 153 Å was set with the coordinates of -0.697, -52.750, -23.233 as x, y, z, respectively, for the EGFR kinase domain protein, and with the same size at -18.081, -43.125, -13.177 as x, y, z, respectively, for the TrkA protein. Simulations were carried out using 100 Lamarckian Genetic Algorithm runs with default parameters. Conformations with the lowest free energy of binding and the most populated cluster were selected for further analysis. Analyses of the interactions were carried out using BIOVIA^® Discovery Studio^® (version 16.1).

To study the antibacterial activity of the sponge-AgNPs mixtures, bacterial cells were obtained from hydatid cyst fluid sourced from various affected anatomical sites, such as the liver and lung, and five different isolated bacterial species were obtained in a previous study by Al Qaisi et al (55). The bacterial species used in the present study were Staphylococcus xylosus, Klebsiella oxytoca, Enterobacter aerogenes, Micrococcus spp. and Escherichia coli. Pseudomonas aeruginosa (cat. no. 27853; ATCC) was used as a control. The isolates were preserved on nutrient agar slants at 4˚C for up to 1 month.

Isolated bacteria were first cultured in Muller Hinton Broth at 37˚C for 24 h for activation before carrying out the antibacterial tests. The antibacterial activity of the sponge extracts and AgNPs were measured using Muller Hinton agar and the disc diffusion method. Inhibitory zone diameters were calculated in mm (56). Briefly, 20 µg of sponge extract (either from Stylissa carteri or Amphimedon chloros) or AgNPs were used to impregnate sterile filter paper discs, which were then put on inoculated Petri dishes containing 0.1 ml of a bacterial solution containing 1.5x10⁸ colony forming units/ml. Discs pre-dosed with 50 g/ml chloramphenicol and 50 g/ml ampicillin were used as positive controls, while discs containing 5% DMSO served as negative controls. After 24 h of incubation at 37˚C, the diameters of the inhibition zones around the extract-impregnated discs were measured and compared with those of the controls. To identify active and inactive sponge preparations, the inhibition zone diameter was used as a metric. Each sample was tested three times. To test for sponge extract-AgNP synergy, two different concentrations were used, which were 10 µg sponge extract and 10 µg AgNPs/disc, and 10 µg sponge extract and 5 µg AgNPs/disc (57).

Results from three or four independent experiments are presented as the means ± standard deviation. Statistical variances between the control group and various treatment groups were evaluated using GraphPad Prism version 10 (Dotmatics). Results were analyzed using one-way ANOVA followed by the Dunnett's test, or using an unpaired t-test. P<0.05 was considered to indicate a statistically significant difference.

A significant difference in charge, but not in size and polydispersity index (PDI) between the pre-and post-lyophilization states are presented in Fig. 1. Before lyophilization, the mean charge was-25.30±0.28 mV, the mean size was 108.65±3.45 nm and the PDI was 0.27±0.07. After the process of lyophilization, the mean size increased to 171.60±13.01 nm, the mean charge increased to -15.05±0.01 mV and the PDI increased to 0.34±0.01. Although all measurements for nanoparticle size (ideal range, 50-200 nm), charge (ideal range, -10 to -30 mV) and PDI (ideal range, ≤0.5) remained within the ideal ranges, these differences indicated that the lyophilization process did not significantly affect the properties of the AgNPs.

SEM analysis reveals uniform distribution and nanoscale size of the AgNPs with minimal aggregation. To investigate the produced AgNPs, SEM was used (Fig. 2). The AgNPs had a uniform distribution and an approximately spherical shape, with negligible aggregation, which indicated a stable production process. Based on the 200 nm scale bar, the majority of the particles had a diameter of <100 nm, which indicated that the size distribution was in the nanoscale zone. Particle aggregation occurs frequently in nanoparticle samples (58); however, in the present study, only minimal aggregation was observed, which indicated high dispersion stability. The AgNPs appeared to have a smooth surface, which is consistent with the nature of nanoparticles made using chemical reduction techniques (59,60). These features implied that these AgNPs were suitable for various applications including anticancer and antimicrobial activity.

To investigate the selective cytotoxicity of AgNPs on the HUVEC normal cell line, an MTT assay was carried out. Cytotoxicity was observed across concentrations ranging from 0.75-200 µg/ml of AgNPs (Fig. 3A). There was a significant difference in the cell cytotoxicity between the 0.75 and 3 µg/ml AgNPs (Fig. 3A), which indicated that 3 µg/ml AgNPs had a lower cytotoxicity compared with 0.75 µg/ml AgNPs in the HUVEC normal cell line. However, 0.75 µg/ml AgNPs were used in the subsequent experiments based on several factors, such as to prevent drug resistance. An advantage of combination therapy is the prevention of drug resistance, which often occurs when high doses of a single agent are used repeatedly (61); therefore, using a lower concentration (0.75 µg/ml) of AgNPs avoided a high-dose exposure, which could otherwise lead to resistance. Additionally, lower doses of nanoparticles may be preferable for long-term use in order to minimize potential side effects while still retaining efficacy (62). Therefore, the subsequent combination experiments used Stylissa carteri or Amphimedon chloros extracts with 0.75 µg/ml AgNPs to minimize the cytotoxic effect of the AgNPs on the HUVEC normal cell line while maximizing the cytotoxic potential against various cancer cell lines. Fig. 3B presents the cytotoxic effects of Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs. Fig. 3C presents the cytotoxicity of Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs. The results indicated a significant reduction in HUVEC cytotoxicity when the Amphimedon chloros extract was combined with AgNPs compared with the Amphimedon chloros extract alone.

Fig. 4A demonstrates the cytotoxic impact of the AgNPs on the A549 cell line. The results revealed a significant increase in the cytotoxicity within the concentration range of 1.5-200 µg/ml AgNPs compared with the concentration of 0.75 µg/ml AgNPs. Fig. 4B presents the cytotoxicity of the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs. Fig. 4C presents the cytotoxic effects of the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs. The results revealed a significant augmentation in the A549 cell cytotoxicity when the Stylissa carteri extract was co-administered with 0.75 µg/ml AgNPs compared with the cytotoxic effects of the Stylissa carteri extract alone.

Fig. 5A demonstrates the cytotoxic effects of the AgNPs on the MCF7 cell line. The results revealed a significant increase in the cytotoxicity within the concentration range of 1.5-200 µg/ml AgNPs compared with the concentration of 0.75 µg/ml AgNPs. Fig. 5B presents the cytotoxicity of the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs, and Fig. 5C presents the cytotoxicity of the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs, when using the MCF7 cell line. However, the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extract with 0.75 µg/ml AgNPs were not significantly different using the MCF7 cell line compared with the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extracts alone, respectively.

Using the PANC-1 cell line, there was a significant increase in the cytotoxicity within the concentration range of 1.5-200 µg/ml AgNPs compared with the concentration of 0.75 µg/ml AgNPs (Fig. 6A). Fig. 6B and C presents the cytotoxic effects of the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs, along with the cytotoxicity of the Amphimedon chloros extract alone and with 0.75 µg/ml AgNPs, when using the PANC-1 cell line. However, the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extract with 0.75 µg/ml AgNPs were not significantly different using the PANC-1 cell line compared with the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extracts alone, respectively.

Fig. 7A presents the cytotoxic effects of the AgNPs on the HT-29 cell line across a concentration range of 0.75-200 µg/ml. The results revealed a significant increase in the cytotoxicity within the concentration range of 1.5-200 µg/ml AgNPs compared with the concentration of 0.75 µg/ml AgNPs. Furthermore, a notable increase in the cell cytotoxicity was observed at AgNP concentrations of 12-200 µg/ml. Additionally, Fig. 7B presents the cytotoxicity of the Stylissa carteri extract alone and in combination with 0.75 µg/ml AgNPs, and Fig. 7C presents the cytotoxicity of the Amphimedon chloros extract alone and in combination with 0.75 µg/ml AgNPs, when using the HT-29 cell line. However, the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extract with 0.75 µg/ml AgNPs were not significantly different using the HT-29 cell line compared with the cytotoxic effects of the Stylissa carteri or the Amphimedon chloros extracts alone, respectively.

LC-MS analysis revealed that there were different chemical components contained in the Stylissa carteri and Amphimedon chloros extracts (Tables I and II, respectively). Based on the LC-MS analysis, manzacidine A, debromohymenialdisine and hymenialdisine were identified as the major components in the Stylissa carteri extract (Fig. 8). Whereas in the Amphimedon chloros extract, methoxyhexadecanoic acid, keramaphidin B and hydroxytricosanoic acid were identified as the major components (Fig. 9). The chemical structures of the predicated components contained in the Stylissa carteri and the Amphimedon chloros extracts are presented in Fig. 10.

The present study investigated the docking interactions of six marine compounds (debromohymenialdisine, hydroxytricosanoic acid, hymenialdisine, keramaphidin B, manzacidine A and methoxyhexadecanoic acid) with the kinase domains of EGFR and TrkA, which have been previously revealed to be overexpressed in different cancer cells such as A549, HT-29, MCF7 and PANC-1 (63-68). The binding energies, specific amino acid interactions, presence of ionic bonds, hydrogen bonds and halogens were analyzed (Table III; Figs. 11 and 12).

All compounds except keramaphidin B formed hydrogen bonds with key amino acid residues such as Leu718, Lys745 or Met793 within EGFR. The results indicated that keramaphidin B had the most favorable binding energy (-7.15 kcal/mol), followed by debromohymenialdisine (-6.68 kcal/mol) and hymenialdisine (-6.65 kcal/mol). Dacomitinib, the control compound, indicated a binding energy of -7.48 kcal/mol and formed hydrogen bonds with Thr790 and Met793. However, it lacked several interactions observed in the marine compounds, such as interactions with Leu718 and Lys745.

Furthermore, keramaphidin B also demonstrated the strongest binding affinity (-7.08 kcal/mol), followed by hymenialdisine (-6.95 kcal/mol) and debromohymenialdisine (-6.71 kcal/mol), against TrkA. Similar to EGFR, all compounds except keramaphidin B formed hydrogen bonds with residues such as Leu516 and Val524.

The findings suggested that several marine compounds, particularly hymenialdisine and debromohymenialdisine, may potentially interact with and inhibit both EGFR and TrkA. These results indicated that these compounds interact with specific residues (Leu718 and Lys745 in EGFR; Leu516 and Val524 in TrkA), which was not observed with the control compounds. Therefore, this may indicate a unique binding mode.

Sponge ethyl acetate extracts combined with AgNPs were tested against six bacterial species using the disc diffusion method (Table IV). In addition, 20 µg/disc AgNPs or sponge ethyl acetate extract alone was administered. The AgNPs and sponge ethyl acetate extract were combined in proportions of 5 or 10 µg/disc of AgNPs with 10 µg/disc of ethyl acetate extract for synergistic tests. Using 20 µg/disc AgNPs alone resulted in ~10 mm-diameter inhibition zones against the majority of bacteria, except for Enterobacter aerogenes, which had an increased susceptibility and an inhibition zone of 28.33 mm. All bacterial species, with inhibition zone diameters ranging from 21.43-13.58 mm, were suppressed by the 20 µg/disc Stylissa carteri ethyl acetate extract. However, the 20 µg/disc Amphimedon chloros extract had no effect on Enterobacter aerogenes or Klebsiella oxytoca, and the remaining bacterial species had inhibition zones that ranged in diameter from 7.34-15.15 mm. When using the extracts alone, the Stylissa carteri extract had an increased inhibitory effect against the six investigated bacterial species compared with the Amphimedon chloros extract. Additionally, when combining 10 µg/disc sponge extract with 5 or 10 µg/disc AgNPs, the majority of the bacterial species investigated had increased inhibition zones and increased fold area (IFA) values when using the Stylissa carteri extract compared with the Amphimedon chloros extract, except for E. coli in which the opposite results were demonstrated. The results indicated that the bacterial species exhibited different responses, which were caused either by the combined treatment of AgNPs with the sponge extract or by the effect of each treatment alone. These results suggest the possibility of using AgNPs in combination with a sponge ethyl acetate extract to treat bacterial infections in hydatid cysts.